AOAC Final Action Methods in 2019

Table 2016.13B. Interlaboratory study results for determination of total lutein a Sample No. of labs used No. of outlier labs b

Mean s r c

s

RSD r

RSD R

HorRat

d

R

NIST SRM 1869

10 10

0 0 1 0 0 2

31.3 0.9 5.2 30.5 2.0 5.3

3.0 6.6

16.6 17.5 11.5 17.0 19.9 12.3

0.87 0.91 0.72 0.78 0.83 0.81

Milk-based toddler formula powder

Adult nutritional powder

9

100.4 2.2 11.5 2.2 12.8 1.5 2.2 12.0

Milk-based infant formula powder 1 Milk-based infant formula powder 2 Chocolate pediatric nutritional RTF

10 10

6.7

0.7 1.3

9.8

8

142.9 3.6 17.6 2.5

a  Results expressed as µg/100 g reconstituted final product. b  Values from outlier laboratories were not included in the statistical analysis. c  s r = SD for repeatability. d  s R = SD for reproducibility.

( q )  Pyrogallol solution (0.2 M pyrogallic acid in reagent alcohol).— Dissolve 6.3 g pyrogallic acid in 250 mL ROH. Store in refrigerator up to 1 month. Solution should be clear at room temperature; discard if colored. ( r )  Extraction solution [1 mM vitamin E in MTBE–THF (1 + 1)].— Combine 50 mL THF/vitamin E solution, C ( p ), with 250 mL MTBE and 200 mL THF. Store in refrigerator up to 1 month. ( s )  Sample solution (10 mM vitamin E in IPA).— Dissolve 3.3 g α-tocopherol in 750 mL IPA. Store in refrigerator for up to 1 month. ( t )  Mobile phase for LC system .—( 1 ) Phase A .—20 mM ammonium acetate in methanol–water (98 + 2). Combine 980 mL MeOH, 20.0 mL water, and 1.54 g ammonium acetate and mix to dissolve. (2) Phase B .—MTBE. D. Standards ( a )  Lutein .—ChromaDex (Part No. ASB-00012453; Irvine, CA, USA), or equivalent. ( b )  β-Carotene .—USP (Part No. 1065480; Rockville, MD, USA), or equivalent. ( c )  Apocarotenal (β-apo-8′-carotenal) .—USP Part No. 1040854, or equivalent. ( d )  Lycopene.— >90% by UV-Vis; Sigma-Aldrich (St. Louis, MO, USA) Part No. L9879, Extrasynthese (Genay, France) Part No. 0305 S, or equivalent.

C. Reagents Note : Reagent volumes may be scaled up or down provided good laboratory practices are followed.

( a )  Laboratory water .—>18 MΩ∙cm. ( b )  Methanol (MeOH) .—HPLC grade. ( c )  MTBE .—HPLC grade.

( d )  n-Hexane .—HPLC grade. ( e )  Cyclohexane .—HPLC grade. ( f )  Potassium hydroxide (KOH) .—Pellets, ACS grade. ( g )  Ethanol (EtOH) .—Pure, HPLC grade. ( h )  Reagent alcohol (ROH) .—Denatured ethanol, HPLC grade. ( i )  α-Tocopherol (vitamin E) .—Synthetic, >95%;Acros Organics Part No. 428121000, Sigma Part No. T3251, or equivalent. ( j )  Pyrogallic acid (pyrogallol) .—ACS grade. ( l )  IPA .—HPLC grade. ( l )  THF .—99.9%, stabilized with BHT. ( m )  Ammonium acetate .—HPLC grade, 98%. ( n )  KOH solution, 50% (w/w).— Add 50 mL water to a 250 mL beaker. Weigh 50 g KOH and slowly transfer to the beaker under constant stirring. When dissolved and cooled, transfer to a media bottle and store at room temperature up to 6 months. ( o )  MTBE/vitamin E solution (10 mM).— Dissolve 1.1 g α-tocopherol in 250 mLMTBE. Store in refrigerator up to 1 month. ( p )  THF/vitamin E solution (10 mM).— Dissolve 1.1 g α-tocopherol in 250 mLTHF. Store in the refrigerator up to 1 month.

Table 2016.13C. Interlaboratory study results for determination of all- trans β-carotene a Sample No. of labs used No. of outlier labs b Mean s r c s R d RSD r RSD R HorRat NIST SRM 1869 8 2 6.9 0.2 0.5 2.9 7.4 0.31 Milk-based toddler formula powder 8 2 7.7 0.3 0.9 4.2 11.1 0.47 Milk-based infant formula powder 1 9 1 10.3 0.2 1.6 2.0 15.3 0.68 Soy-based infant formula powder 9 1 11.7 0.5 1.4 4.1 11.8 0.53 Milk-based infant formula powder 2 9 1 14.1 0.3 1.8 2.2 12.5 0.58 Infant formula RTF 8 2 15.9 0.3 1.5 2.0 9.4 0.45 a  Results expressed as µg/100 g reconstituted final product. b  Values from outlier laboratories were not included in the statistical analysis. c  s r = SD for repeatability. d  s R = SD for reproducibility.

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